This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We are using HDX and FPOP to probe the interface of insulin oligomers. Research indicates that specific residues on insulin play the key role in oligomerization and different residues are involved in various oligomers. For example, if LysB28 and ProB29 are reversed, dimerization constant will decrease drastically;if GluB13 is changed to Gln, the mutants will have higher propensity to form hexamers. Most of the previous research was based on measuring thermodynamic and kinetic data of insulin mutants, which were not only time-consuming but also not able to elucidate global properties. HDX and FPOP provide the opportunities to map out all the interactions involved in oligomerization without using mutants. By adjusting the pH, concentration, and additives, certain oligomers can be dominant in the distribution. With a top-down strategy particular precursors can be selected in the gas-phase and fragmented. We can distinguish the effect of binding for different oligomers. We plan to pinpoint which residues are crucial for binding in specific oligomers.